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MB Sample ID: SA125428
Local Sample ID: | 55_ |
Subject ID: | SU001563 |
Subject Type: | Plant |
Subject Species: | Zea mays |
Taxonomy ID: | 4577 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001563 |
Subject Type: | Plant |
Subject Species: | Zea mays |
Taxonomy ID: | 4577 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
55_ | SA125428 | FL015360 | Control | Sample Type |
55_ | SA125428 | FL015360 | 14 day | Age |
55_ | SA125428 | FL015360 | 38 returned to 38 | Growth Temperature |
Collection:
Collection ID: | CO001558 |
Collection Summary: | Samples were collected and flash frozen in liquid N2 |
Sample Type: | Plant |
Treatment:
Treatment ID: | TR001578 |
Treatment Summary: | For inoculations, single isolates of Cochliobolus heterostrophus were cultured on V8 agar for 3-4 weeks as described by (Christensen et al., 2018). Spot inoculations were performed on 14-d-old plants in which four 10 μl droplets of either C. heterostrophus suspension (1x106 spores mL-1) or control solution (sterile 0.1% Tween 20) were applied in a staggered fashion (Fig. 2a and Fig. 4a) onto the middle portion of the third leaf about 10cm from the distal tip. Following inoculation, both heat-stressed and non-heat-stressed plants were placed in a plastic humidity chamber and placed in the 28°C Percival. At 24 hours post-inoculation, infected and non-infected controls were removed from the humidity chambers and maintained at 28°C for the remainder of the experiment. At 72 hours post-inoculation, leaves (n=6) were photographed and immediately harvested into liquid N2. Lesions were digitally measured using ImageJ software (ImageJ 1.36b; Wayne Rasband, NIH, Bethesda, MD, USA). |
Sample Preparation:
Sampleprep ID: | SP001571 |
Sampleprep Summary: | LCMS grade reagents (Thermo Fisher Scientific) were used throughout the extraction. Frozen 1.5-mL fast prep tubes with Zirmil® beads (1.1mm; SEPR Ceramic Beads and Powders, Mountainside, NJ, USA) containing 50 mg of ground and frozen tissue were carefully thawed to 4°C then transferred to ice. An internal standard mix of 12 μL containing caffeine, D6-Abscisic acid, D5-Jasmonic acid, D5-Cinnamic acid, D5-Indole-3-acetic acid, 13C-alpha linolenic acid, and nicotine at 8.33 μg/mL was added to each sample along with 750 μL of 10 mM of ammonium acetate and 750 μL of methanol (MeOH). Samples were mixed by vortex then homogenized at 6000 RPM for 30 seconds in a FastPrep® FP 120 tissue homogenizer (Qbiogene) and then sonicated in an ambient water bath for 15 min. Samples were centrifuged for 10 minutes at 20,000 RCF at ambient temperature, then 1 mL of supernatant was transferred to individual 4 mL glass vials. The supernatant was dried under a nitrogen stream at 30°C, reconstituted in 200 μL of 0.1% formic acid in water, and vortexed for 30 seconds. Vial contents were transferred to a 1.5-mL snap-cap tube, placed on ice for 10 minutes, then centrifuged at 20,000 RCF for 10 minutes at ambient temperature. 150 μL of supernatant was transferred to glass LC vials for UHPLC-HRMS analysis. |
Combined analysis:
Analysis ID | AN002466 | AN002467 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Thermo Vanquish | Thermo Vanquish |
Column | ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) | ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | peak intensity | peak intensity |
Chromatography:
Chromatography ID: | CH001808 |
Instrument Name: | Thermo Vanquish |
Column Name: | ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS002286 |
Analysis ID: | AN002466 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The raw acquisition data were processed using a similar workflow described in previous work (Chamberlain et al., 2019a, b), which we detail here. Raw data files were converted from .raw to .mzxml format using RawConverter (He et al., 2015). MZmine 2 was used for processing the raw data including detecting masses, building chromatograms, grouping isotopic peaks, removing duplicate peaks, and aligning features (Pluskal et al., 2010). Identification was assigned to features by m/z (≤5 ppm) and retention time (0.2 min) (level 1 – identified compounds according to Metabolomics Standards Initiative standards (Sumner et al., 2007)) matching tousing our method-specific metabolite library produced from pure standards previously analyzed using this the above-mentioned chromatographic gradient. Processed data were exported from MZmine as a feature list containing the signal intensity for each feature in each sample. A small value (half the minimum value in the dataset) was used to replace zeros (no detection). The data were filtered to remove sample features with 10% signal contribution from their corresponding features in the extraction blanks. From this point, the data were further processed, normalized, and filtered using MetaboAnalyst 4.0 (Chong et al., 2018). For whole-metabolome comparative analyses, the data were normalized to total ion signal and feature intensities were auto-scaled to facilitate statistical comparisons (van den Berg et al., 2006). Statistical significance, defined as p 0.05, was determined using the two-tailed student’s t-test, and values for significance were adjusted for the false discovery rate with the Bonferroni-Holm method (HOLM, 1979). |
Ion Mode: | POSITIVE |
MS ID: | MS002287 |
Analysis ID: | AN002467 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The raw acquisition data were processed using a similar workflow described in previous work (Chamberlain et al., 2019a, b), which we detail here. Raw data files were converted from .raw to .mzxml format using RawConverter (He et al., 2015). MZmine 2 was used for processing the raw data including detecting masses, building chromatograms, grouping isotopic peaks, removing duplicate peaks, and aligning features (Pluskal et al., 2010). Identification was assigned to features by m/z (≤5 ppm) and retention time (0.2 min) (level 1 – identified compounds according to Metabolomics Standards Initiative standards (Sumner et al., 2007)) matching tousing our method-specific metabolite library produced from pure standards previously analyzed using this the above-mentioned chromatographic gradient. Processed data were exported from MZmine as a feature list containing the signal intensity for each feature in each sample. A small value (half the minimum value in the dataset) was used to replace zeros (no detection). The data were filtered to remove sample features with 10% signal contribution from their corresponding features in the extraction blanks. From this point, the data were further processed, normalized, and filtered using MetaboAnalyst 4.0 (Chong et al., 2018). For whole-metabolome comparative analyses, the data were normalized to total ion signal and feature intensities were auto-scaled to facilitate statistical comparisons (van den Berg et al., 2006). Statistical significance, defined as p 0.05, was determined using the two-tailed student’s t-test, and values for significance were adjusted for the false discovery rate with the Bonferroni-Holm method (HOLM, 1979). |
Ion Mode: | NEGATIVE |